Cardiac looping is a vital morphogenetic event during early development. During looping, the relatively straight heart tube bends and twists, moving the future atria and ventricles into their correct relative anatomical positions. Even relatively minor perturbations in looping geometry can lead to major congenital heart defects. Thus, it is important to determine the factors that drive and regulate this process. Several ideas have been proposed for the mechanism of looping, but experimental results have been inconclusive and sometimes conflicting. Thus, cardiac looping remains a poorly understood process. Looping likely involves a dynamic interaction between genetic and epigenetic factors, with biomechanics being a major epigenetic component. The main objective of this research is to investigate the role that biomechanical forces play in the looping process. The specific aims of the proposed research will test the following main hypotheses: (1) Residual stress, in particular longitudinal tension in the cardiac tube near the dorsal mesocardium, plays a role in looping. (2) Looping is driven in part by swelling of the cardiac jelly, coupled with regional variations in stiffness and anisotropy of the myocardium. (3) Cytoskeletal contraction in the myocardium plays a role in looping. These hypotheses will be tested using a combination of experimental and theoretical methods. Experiments conducted on chick embryo hearts will perturb the mechanism studied in each specific aim and measure the effects on looping morphology and the material properties of the cardiac tube. The material properties, measured by "cell poker" and atomic force microscopy, and geometry reconstructed from serial sections will be used to develop computational models for the embryonic heart based on fundamental physical principles. These models will include the effects of large deformation, anisotropy, active contraction, growth, and complex three-dimensional geometry. The models will predict looping geometry for each experimental perturbation, and the actual looping mechanism(s) will be determined by comparing measured and predicted looping morphologies. Defining the biomechanical forces involved in looping would provide insight into this morphogenetic process and thereby help researchers searching for the link between gene expression and looping morphology.